A New Way To Use RNA Interference for Cancer Therapy
By Lara van Rooyen
If you were to inject a brightly colored flower with the RNA (ribonucleic acid) of its pigment, what would happen? This might seem like a strange question—indeed, this question was never asked until 1996, when a team of scientists obtained a seemingly impossible result from an attempt to create flowers with a darker color: when petunia flowers were injected with the RNA of their purple pigment, the flowers developed white patches instead of the expected deeper purple color. It seemed as though the gene for the pigment was inexplicably being suppressed, despite the apparent excess of pigment RNA present [1].
As this puzzling result was further investigated over the next few decades, a similar phenomenon of gene silencing through foreign RNA introduction was found in a range of other organisms. As such, this process was aptly named RNA interference, or RNAi. It was later discovered that RNAi appeared to be mediated by the activation of an enzyme complex called RISC. Upon foreign RNA binding to the RISC complex, the enzyme proceeds to degrade both the self and non-self copies of that specific RNA. In the case of the petunias, both the injected pigment RNA and the endogenous (preexisting) pigment RNA in the cells were destroyed, resulting in flowers devoid of pigmentation.
RNAi has since been used as an efficient way for scientists to ‘turn off’ specific genes, since a gene’s expression depends on the presence of its specific RNA molecules. Following the discovery of RNAi, the focus has shifted to taking advantage of this gene silencing for therapeutic purposes, as a large number of disease states are associated with excessive expression of certain genes. For example, one of mankind’s most feared diseases—cancer—is related to the overexpression of oncogenes. When mutated, these oncogenes overperform their functions, which include cell growth and proliferation, leading to uncontrolled cell division that eventually results in tumor formation [2]. RNAi provides a new hope for cancer patients, as it can target multiple genes without the adverse effects produced by the simultaneous use of several chemotherapeutic drugs and is also able to target biochemical pathways for which no drugs have yet been developed [3]. However, despite the great promise RNAi shows as a highly individualized cancer treatment, it still results in some off-target effects with various other genes suffering unintended alterations in their expression [4]. Another type of off-target effect occurs at the cellular level, when there are inaccuracies in delivery of the foreign RNA.
Research published in 2019 by a team of French scientists proposed a TAG-RNAi system to combat this cellular off-target effect, and this system allows only specific cells to receive the RNAi treatment while all other cells are spared the undesired changes to their gene expression. This is done by adding a tag to the cell’s own RNA and then fusing a sequence to the injected RNA that will allow it to bind to only the host RNA, thereby causing its degradation through RNAi [5]. To demonstrate the efficacy and specificity of their TAG-RNAi system, the team implanted cancer cells into mice. One flank was implanted with ‘tagged’ cancer cells while the cells implanted in the other flank lacked this tag. This allowed for a contralateral control, since many variables could be kept constant due to both control and experimental conditions being present in the same organism. As was hoped for, the tagged tumor cells showed significantly reduced growth and division when RNAi was applied against a specific oncogene while the untagged flank developed a tumor that displayed uninhibited growth. In contrast, untagged cells were not affected by the RNAi, so their functions continued uninterrupted, preventing any systemic side effects.
The recent revolutionary approach of altering the function of genes in order to treat disease has not been limited to RNAi. CRISPR, a gene-editing system that can alter specific nucleotides within the gene sequence, has garnered massive media attention and has also been proposed as a possible cancer treatment. However, while CRISPR-based therapeutics seem promising, the permanent molecular changes that they produce are seen as controversial, especially considering the possibility of off-target gene alterations [6]. Since RNAi provides a way to silence a gene without changing it irreversibly and can now be made more accurate by tagging specific groups of cells, it might soon take the center stage in the ongoing battle against diseases such as cancer.
With the first RNAi-based drug having been approved by the FDA in 2018, clinicians and scientists all around the world are taking notice of the potential of harnessing this system [7]. As a result of this new method of improving RNAi specificity through the TAG-RNAi system, we might soon see the dozens of current clinical trials involving RNAi come to fruition. From unexpected results in an experiment on petunias to a groundbreaking new clinical therapy in only a few decades, RNAi truly showcases the human drive to adapt discoveries to our needs—who knows what we will be able to do with RNAi in just a few decades more.
References
If you were to inject a brightly colored flower with the RNA (ribonucleic acid) of its pigment, what would happen? This might seem like a strange question—indeed, this question was never asked until 1996, when a team of scientists obtained a seemingly impossible result from an attempt to create flowers with a darker color: when petunia flowers were injected with the RNA of their purple pigment, the flowers developed white patches instead of the expected deeper purple color. It seemed as though the gene for the pigment was inexplicably being suppressed, despite the apparent excess of pigment RNA present [1].
As this puzzling result was further investigated over the next few decades, a similar phenomenon of gene silencing through foreign RNA introduction was found in a range of other organisms. As such, this process was aptly named RNA interference, or RNAi. It was later discovered that RNAi appeared to be mediated by the activation of an enzyme complex called RISC. Upon foreign RNA binding to the RISC complex, the enzyme proceeds to degrade both the self and non-self copies of that specific RNA. In the case of the petunias, both the injected pigment RNA and the endogenous (preexisting) pigment RNA in the cells were destroyed, resulting in flowers devoid of pigmentation.
RNAi has since been used as an efficient way for scientists to ‘turn off’ specific genes, since a gene’s expression depends on the presence of its specific RNA molecules. Following the discovery of RNAi, the focus has shifted to taking advantage of this gene silencing for therapeutic purposes, as a large number of disease states are associated with excessive expression of certain genes. For example, one of mankind’s most feared diseases—cancer—is related to the overexpression of oncogenes. When mutated, these oncogenes overperform their functions, which include cell growth and proliferation, leading to uncontrolled cell division that eventually results in tumor formation [2]. RNAi provides a new hope for cancer patients, as it can target multiple genes without the adverse effects produced by the simultaneous use of several chemotherapeutic drugs and is also able to target biochemical pathways for which no drugs have yet been developed [3]. However, despite the great promise RNAi shows as a highly individualized cancer treatment, it still results in some off-target effects with various other genes suffering unintended alterations in their expression [4]. Another type of off-target effect occurs at the cellular level, when there are inaccuracies in delivery of the foreign RNA.
Research published in 2019 by a team of French scientists proposed a TAG-RNAi system to combat this cellular off-target effect, and this system allows only specific cells to receive the RNAi treatment while all other cells are spared the undesired changes to their gene expression. This is done by adding a tag to the cell’s own RNA and then fusing a sequence to the injected RNA that will allow it to bind to only the host RNA, thereby causing its degradation through RNAi [5]. To demonstrate the efficacy and specificity of their TAG-RNAi system, the team implanted cancer cells into mice. One flank was implanted with ‘tagged’ cancer cells while the cells implanted in the other flank lacked this tag. This allowed for a contralateral control, since many variables could be kept constant due to both control and experimental conditions being present in the same organism. As was hoped for, the tagged tumor cells showed significantly reduced growth and division when RNAi was applied against a specific oncogene while the untagged flank developed a tumor that displayed uninhibited growth. In contrast, untagged cells were not affected by the RNAi, so their functions continued uninterrupted, preventing any systemic side effects.
The recent revolutionary approach of altering the function of genes in order to treat disease has not been limited to RNAi. CRISPR, a gene-editing system that can alter specific nucleotides within the gene sequence, has garnered massive media attention and has also been proposed as a possible cancer treatment. However, while CRISPR-based therapeutics seem promising, the permanent molecular changes that they produce are seen as controversial, especially considering the possibility of off-target gene alterations [6]. Since RNAi provides a way to silence a gene without changing it irreversibly and can now be made more accurate by tagging specific groups of cells, it might soon take the center stage in the ongoing battle against diseases such as cancer.
With the first RNAi-based drug having been approved by the FDA in 2018, clinicians and scientists all around the world are taking notice of the potential of harnessing this system [7]. As a result of this new method of improving RNAi specificity through the TAG-RNAi system, we might soon see the dozens of current clinical trials involving RNAi come to fruition. From unexpected results in an experiment on petunias to a groundbreaking new clinical therapy in only a few decades, RNAi truly showcases the human drive to adapt discoveries to our needs—who knows what we will be able to do with RNAi in just a few decades more.
References
- Jorgensen, R.A., et al. “Chalcone Synthase Cosuppression Phenotypes in Petunia Flowers: Comparison of Sense vs. Antisense Constructs and Single-Copy vs. Complex T-DNA Sequences.” Plant Molecular Biology, no. 5, 1996, pp. 957–973.
- Croce, Carlo M. “Oncogenes and Cancer.” The New England Journal of Medicine, vol. 358, no. 5, 2008, pp. 502–511.
- Wu, Sherry Y, et al. “RNAi Therapies: Drugging the Undruggable.” Science Translational Medicine, vol. 6, no. 240, 2014, p. 240ps7.
- Klitgord, Niels, et al. “Novel Insights into RNAi Off-Target Effects Using C. Elegans Paralogs.” BMC Genomics, vol. 8, no. 1, 2007, p. 106.
- Champagne, Julien, et al. “TAG-RNAi Overcomes off-Target Effects in Cancer Models.” Oncogene, 2019, pp. Oncogene, September 26, 2019.
- Zhou, Changyang, et al. “Off-Target RNA Mutation Induced by DNA Base Editing and Its Elimination by Mutagenesis.” Nature, vol. 571, no. 7764, 2019, pp. 275–278.
- Setten, Ryan L, et al. “The Current State and Future Directions of RNAi-Based Therapeutics.” Nature Reviews. Drug Discovery, vol. 18, no. 6, 2019, pp. 421–446.
- S. Mocellin, M. Provenzano, Wikimedia Commons, Wikimedia Commons, 2007. https://commons.wikimedia.org/wiki/File:Mechanism_of_RNA_interference.jpg (accessed January 27, 2020)
Figure 1. Mechanism of RNA Interference [8]
